A number of successful protein therapeutics are recombinant fusion proteins consisting of two proteins or protein domains fused together through a linker, or a protein scaffold into which one or more domains from a second protein have been grafted. Typically, such fusion proteins are designed to leverage beneficial properties of each member of the fusion.
For example, cytokines or growth factors have been fused with the Fc portion of IgG1 or immunotoxin and expressed as single polypeptides with dual biological activities. Examples of therapeutic fusion proteins that have been developed using cytokines or growth factors and the Fc portion of IgG1 include Enbrel® (TNF-RIFs-IgG1), Ontak® (IL-2/diphtheria toxin), Orencia® (CTLA-4/Fc-IgG1) and Amevive® (LFA-3/Fc-IgG1).
Protein engineering has been used extensively to introduce novel binding specificities into protein scaffolds. Both rational and combinatorial approaches have been used with a variety of structurally diverse scaffolds (see Binz et al., 2005, Nature Biotechnology, 23(10):1257-1268; Nygren & Skerra (2004, J Immunol. Methods, 290:3-28) and Gebauer & Skerra (2009, Curr. Op. Chem. Biol., 13:245-255). Antibodies are perhaps the best studied of all protein scaffolds and affinity transfer by loop swapping has become routine. The technique of loop swapping was first described by Jones et al. (1986, Nature 321(6069):522-525), who substituted the CDRs from the heavy chain variable region of a mouse antibody, which binds to the hapten 4-hydroxy-3-nitrophenacetyl caproic acid (NP-cap), for the corresponding CDRs of a human myeloma antibody. It is now quite common to transfer the complementarity determining region (CDR) loops from a non-human antibody to the scaffold of a human antibody to increase its therapeutic potential (Jones et al., 1986, ibid; Riechmann et al., 1988, Nature 332(6162):323-327; Verhoeyen et al., 1988, Science 239(4847):1534-1536).
Affinity transfer by CDR replacement has also been successful with non-immunoglobulin scaffolds. Nicaise et al. (2004, Protein Sci 13(7):1882-1891) grafted the CDR3 of a lysozyme-specific camel antibody onto neocarzinostatin (NCS). Novel binding properties have also been generated by transferring CDR-like loops from proteins other than antibodies, for example, van den Beucken et al. (2001, J Mol Biol 310(3):591-601) made a VL library with a constant CDR3-like sequence from the protein CLTA-4, and selected variants with specificity for its receptor B7.1 and demonstrated that the flanking conformational context is important in maintaining functional binding properties of the transferred domain. Several non-antibody scaffolds are also being evaluated for use as potential therapeutics including fibronectin (Hackel et al., 2008, J Mol Biol 381(5):1238-1252; Lipovsek et al., 2007, J Mol Biol 368(4):1024-1041), lipocalins, avimers, adnectins and ankyrins. Zeytun et al. (2003, Nat Biotechnol 21(12):1473-1479), introduced diverse CDR-H3 sequences into four surface loops of an optimised GFP scaffold to create “fluorobodies.”
Various methods have been used to introduce diversity into these scaffolds including error prone PCR approaches, degenerate oligo or peptide synthesis or a variety of DNA/CDR shuffling and CDR walking strategies (Bernath et al., 2005, J Mol Biol 345(5):1015-1026; Nord et al., 1997, Nat Biotechnol 15(8):772-777; Colas et al., 1996, Nature 380(6574):548-550).
The first report of peptide being placed into the CDR of an antibody was by Sallazzo (1990). Placing peptides into a CDR of antibody and maintaining peptide function is often compromised because the peptide is no longer unconstrained or is constrained in an inappropriate confirmation. Successful insertion of RGD peptides into the CDR3 of the antibody heavy chain has been reported (Zanetti et al., 1993, EMBO J, 12(11):4375-4384). Simon et al. (2005, Arch Biochem Biophys, 440(2):148-157) describes the insertion of the somatostatin peptide into the CDRs of the kappa light chain using PCR mediated gene splicing by overlap extension. The points of insertion were identified through alignment of kappa light chain variable region amino acid sequences and X-ray crystal structures. The authors confirmed that somatostatin peptides inserted into the predicted regions of kappa CDR-1 and CDR-2 were able to bind to membranes containing somatostatin receptor 5.
A TPO agonist antibody has also been described that utilized insertion of two copies of an active peptide into CDR loops of an antibody fragment (Fab) (Frederickson et al., 2006, PNAS USA, 103(39):14307-14312). The group reported that the amino acids flanking the peptide required optimization for proper presentation of the peptide in the context of the antibody scaffold. Using phage display, two amino acids on either side of the peptide were randomized and inserted in to CDR3 of the heavy chain and subsequent panning identified binders. Several of the identified binders also showed agonist activity.
V(D)J recombination is the process responsible of the assembly of antibody gene segments (V, D and J; or V and J in the case of the light chain) and as part of the assembly process creates the CDR3 of the respective antibody chain. V(D)J recombination can be considered conceptually as a segment shuffler for antibodies, i.e. it brings together the different VH segments, D segments and JH segments to create an antibody (similarly V(D)J recombination at the light chain assembles different combinations of light chain V and J segments at either the kappa or lambda locus). The recombination event results in large chromosomal deletions in order to bring the required segments together. V(D)J recombination is targeted by the presence of specific DNA sequences called the recombination signal sequences (RSSs). The recombination reaction involves the recombination proteins RAG-1 and RAG-2 and follows a 12/23 rule where an RSS with a 23 bp spacer is paired only with an RSS with 12 bp spacer and adjacent sequences are subsequently joined by double-stranded break repair proteins.
U.S. Pat. No. 8,012,714 describes compositions and methods for generating sequence diversity in the CDR3 region of de novo generated immunoglobulins in vitro. The methods comprise constructing nucleic acid molecules that comprise polynucleotide sequences encoding immunoglobulin V, D, J and C regions, together with recombination signal sequences (RSS), and subsequently introducing these nucleic acid molecules into suitable recombination-competent host cells.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.